Bacterial DNA replication is carefully controlled at the initiation stage, including by regulation of the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence indicating membranes have a profound influence on DnaA protein. DnaA protein is found at the cell membrane in vivo, and membrane liposomes containing acidic phospholipids can reactivate an inert form of DnaA protein into replicatively active DnaA in vitro, with a distinct region of DnaA protein found inserted into the hydrophobic interior of the membrane bilayer. Conversely, the membrane-associated protein, Hda participates in converting active DnaA into its inactive form commensurate with DNA replication. Certain mutant forms of DnaA protein are able to suppress the arrested growth of cells that lack an adequate level of acidic phospholipids, further implicating a close link between chromosomal replication and the cell membrane. The long-term goal of this research is to elucidate the physiological significance of the influence of membranes on chromosomal replication. The research outlined here uses biochemical approaches with defined components, physiological studies, cytolocalization techniques, and structural studies to directly test the hypothesis that the cellular membrane participates in the regulation of DnaA protein activity. The Specific Aims are to: 1. Analyze how an inadequate level of acidic phospholipids affects the cell-cycle, and identify the molecular mechanism of how DnaA(L366K) suppresses the growth-arrest of acidic phospholipid-deficient cells. Determine whether cells are arrested at a specific point or randomly in the cell-cycle as they are depleted of acidic phospholipids. Identify the properties or functions of DnaA(L366K), distinct from wild-type DnaA, that allow continued growth in cells lacking sufficient amounts of acidic phospholipids. 2. Determine if acidic phospholipids participate in the cell-cycle localization of DnaA and the chromosomal origin, and the membrane association of Hda protein. Using fluorescence microscopy of living cells, examine the importance of acidic phospholipids in the proper localization of DnaA, the chromosomal origin, and the DnaA-regulating factor, Hda, as cells progress through the cell-cycle. 3. Define the structure of DnaA protein bound to acidic lipid bilayers. Use the lipidic cubic phase in meso method to obtain diffraction-quality crystals for high-resolution structural determination of DnaA protein associated with a lipid bilayer that contains acidic phospholipids. This work should provide insight into the interactions between membrane lipids and DNA synthesis components, and the functions that membranes may play in cell-cycle and growth controlled initiation of chromosomal replication. Furthermore, advances in understanding of how membrane lipids influence DnaA may help direct future investigations in the emerging field of phospholipid-mediated regulation of enzymatic activities. [unreadable] [unreadable] [unreadable]